Biomarkers of therapeutic responsiveness

ABSTRACT

The present invention relates to methods of diagnosing breast cancer in a patient, as well as methods of monitoring the progression of breast cancer and/or methods of monitoring a treatment protocol of a therapeutic agent or a therapeutic regimen. The invention also relates to assay methods used in connection with the diagnostic methods described herein.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of co-pending application Ser.No. 14/168,629, filed Jan. 30, 2014, which claims priority of U.S.Provisional Application No. 61/759,431 filed on Feb. 1, 2013, the entirecontents of which are incorporated herein by reference.

STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH

This invention was made with federal support under HHSN261201000075Cawarded by the National Cancer Institute. The U.S. government hascertain rights in the invention.

FIELD OF THE INVENTION

This application relates to assay methods useful in the detection andtreatment of breast cancer.

BACKGROUND OF THE INVENTION

Challenges in the field of oncology include the lack of efficient meansfor early cancer detection and for specific cancer subtyping and formeasuring and/or predicting responsiveness to therapy. There is a needfor new cancer biomarkers that can provide early and specific diagnosisof cancer and enable targeted therapy and prognosis. The need for newdiagnostics has been the impetus behind many initiatives targeting thediscovery and development of new biomarkers for cancer. The hope is thatthe identification of suitable biomarkers will allow for the developmentof early cancer detection screening tests and will lead to improvedcancer therapy and a reduction in the mortality associated with manycancers.

SUMMARY OF THE INVENTION

The invention provides a method for evaluating the efficacy of atreatment regimen in a patient diagnosed with breast cancer, said methodcomprising

(a) obtaining a test sample from a patient undergoing said treatmentregimen for breast cancer;

(b) measuring a level of a biomarker in said test sample, wherein saidbiomarker comprises (i) total and phosphorylated isoforms of Akt, MEK,mTOR, GSK3beta, and combinations thereof, (ii) p70S6K, eIF4EBP1, PTEN,and (iii) combinations thereof;

(c) comparing said level to a normal control level of said biomarker;and

(d) evaluating from said comparing step (c) whether said patient isresponsive to said treatment regimen.

The invention further provides a method for evaluating the efficacy of atreatment regimen in a patient diagnosed with breast cancer, said methodcomprising

(a) ordering a test comprising a measurement of a level of a biomarkerin a test sample obtained from a patient undergoing said treatmentregimen for breast cancer, wherein said biomarker comprises (i) totaland phosphorylated isoforms of Akt, MEK, mTOR, GSK3beta, andcombinations thereof, (ii) p70S6K, eIF4EBP1, PTEN, and (iii)combinations thereof;

(b) comparing said level to a normal control level of said biomarker;and

(c) evaluating from said comparing step (b) whether said patient isresponsive to said treatment regimen.

Another embodiment of the invention is a method of administering atreatment regimen to a patient in need thereof for treating breastcancer, comprising:

(a) obtaining a test sample from a patient undergoing said treatmentregimen for breast cancer;

(b) measuring a level of a biomarker in said test sample, wherein saidbiomarker comprises (i) total and phosphorylated isoforms of Akt, MEK,mTOR, GSK3beta, and combinations thereof, (ii) p70S6K, eIF4EBP1, PTEN,and (iii) combinations thereof;

(c) comparing said level to a normal control level of said biomarker;

(d) evaluating from said comparing step (c) whether said patient isresponsive to said treatment regimen; and

(e) adjusting said treatment regimen based on said evaluating step (d).

Still further, the invention contemplates a method of administering atreatment regimen to a patient in need thereof for treating breastcancer, comprising:

(a) obtaining a test sample from a patient prior to the commencement ofsaid treatment regimen for breast cancer;

(b) measuring a level of a biomarker in said test sample, wherein saidbiomarker comprises (i) total and phosphorylated isoforms of Akt, MEK,mTOR, GSK3beta, and combinations thereof, (ii) p70S6K, eIF4EBP1, PTEN,and (iii) combinations thereof;

(c) comparing said level to a normal control level of said biomarker;

(d) evaluating from said comparing step (c) whether said patient will beresponsive to said treatment regimen; and

(e) administering said treatment regimen based on said evaluating step(d).

An alternative or additional embodiment of the invention is a method ofadministering a treatment regimen to a patient in need thereof fortreating breast cancer, comprising:

(a) evaluating a level of a biomarker in a test sample obtained from apatient undergoing said treatment regimen for breast cancer relative toa normal control level of said biomarker, wherein said biomarkercomprises (i) total and phosphorylated isoforms of Akt, MEK, mTOR,GSK3beta, and combinations thereof, (ii) p70S6K, eIF4EBP1, PTEN, and(iii) combinations thereof; and

(b) adjusting said treatment regimen based on said evaluating step (a).

Moreover, the invention includes a method of administering a treatmentregimen to a patient in need thereof for treating breast cancer,comprising:

(a) evaluating a level of a biomarker in a test sample obtained from apatient prior to the commencement of said treatment regimen for breastcancer relative to a normal control level of said biomarker, whereinsaid biomarker comprises (i) total and phosphorylated isoforms of Akt,MEK, mTOR, GSK3beta, and combinations thereof, (ii) p70S6K, eIF4EBP1,PTEN, and (iii) combinations thereof; and

(b) administering said treatment regimen based on said evaluating step(a).

The invention also provides a kit for the analysis of a breast cancerpanel comprising

(a) a multi-well assay plate comprising a plurality of wells, each wellcomprising at least four discrete binding domains to which captureantibodies to the following human analytes are bound: (i) total andphosphorylated isoforms of Akt, MEK, mTOR, GSK3beta, and combinationsthereof, (ii) p70S6K, eIF4EBP1, PTEN, and (iii) combinations thereof;

(b) in one or more vials, containers, or compartments, a set of labeleddetection antibodies specific for said human analytes; and

(c) in one or more vials, containers, or compartments, a set ofcalibrator proteins.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the fractional marker levels (to 0 h time point) for arepresentative set of biomarkers.

FIGS. 2(a)-(d) show the relative levels of optimal biomarkers as afunction of IC50 values for the 1 hour time point (cut off forsensitivity is 1 uM). Each point on the graphs represents a cell line.

FIG. 3 shows the relative levels of the combined markers phospho-Akt,phospho-GSK3beta, phospho-MEK, and phospho-mTOR as a function of IC50values for the 1 hour time point. Each point on the graph represents thesum of the relative levels of each marker shown in FIG. 2(a)-(d) for onecell line as a function of its 1050 value for gefitinib.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. The articles“a” and “an” are used herein to refer to one or to more than one (i.e.,to at least one) of the grammatical object of the article. By way ofexample, “an element” means one element or more than one element.

As used herein, the term “sample” is intended to mean any biologicalfluid, cell, tissue, organ or combinations or portions thereof, whichincludes or potentially includes a biomarker of a disease of interest.For example, a sample can be a histologic section of a specimen obtainedby biopsy, or cells that are placed in or adapted to tissue culture. Asample further can be a subcellular fraction or extract, or a crude orsubstantially pure nucleic acid molecule or protein preparation. In oneembodiment, the samples that are analyzed in the assays of the presentinvention are blood, peripheral blood mononuclear cells (PBMC), isolatedblood cells, serum and plasma. Other suitable samples include biopsytissue, intestinal mucosa, saliva, cerebral spinal fluid, and urine.

A “biomarker” is a substance that is associated with a particulardisease. A change in the levels of a biomarker may correlate with therisk or progression of a disease or with the susceptibility of thedisease to a given treatment. A biomarker may be useful in the diagnosisof disease risk or the presence of disease in an individual, or totailor treatments for the disease in an individual (choices of drugtreatment or administration regimes and/or to predict responsiveness ornon-responsiveness to a particular therapeutic regimen). In evaluatingpotential drug therapies, a biomarker may be used as a surrogate for anatural endpoint such as survival or irreversible morbidity. If atreatment alters a biomarker that has a direct connection to improvedhealth, the biomarker serves as a “surrogate endpoint” for evaluatingclinical benefit. A sample that is assayed in the diagnostic methods ofthe present invention may be obtained from any suitable patient,including but not limited to a patient suspected of having breast canceror a patient having a predisposition to breast cancer. The patient mayor may not exhibit symptoms associated with one or more of theseconditions.

“Level” refers to the amount, concentration, or activity of a biomarker.The term “level” may also refer to the rate of change of the amount,concentration or activity of a biomarker. A level can be represented,for example, by the amount or synthesis rate of messenger RNA (mRNA)encoded by a gene, the amount or synthesis rate of polypeptidecorresponding to a given amino acid sequence encoded by a gene, or theamount or synthesis rate of a biochemical form of a biomarkeraccumulated in a cell, including, for example, the amount of particularpost-synthetic modifications of a biomarker such as a polypeptide,nucleic acid or small molecule. The term can be used to refer to anabsolute amount of a biomarker in a sample or to a relative amount ofthe biomarker, including amount or concentration determined understeady-state or non-steady-state conditions. Level may also refer to anassay signal that correlates with the amount, concentration, activity orrate of change of a biomarker. The level of a biomarker can bedetermined relative to a control marker.

As used herein, the term “cancer” is intended to mean a class ofdiseases characterized by the uncontrolled growth of aberrant cells,including all known cancers, and neoplastic conditions, whethercharacterized as malignant, benign, soft tissue or solid tumor. Asidefrom non-melanoma skin cancer, breast cancer is the most common canceramong women in the United States. It is also one of the leading causesof cancer death among women of all races and Hispanic originpopulations. About 1 in 8 (12%) women in the US will develop invasivebreast cancer during their lifetime. The American Cancer Societyestimates that in 2013 about 232,340 new cases of invasive breast cancerwill be diagnosed in women, about 64,640 new cases of carcinoma in situ(CIS) will be diagnosed (CIS is non-invasive and is the earliest form ofbreast cancer), and about 39,620 women will die from breast cancer.Breast cancer is the second leading cause of cancer death in women,second only to lung cancer. The chance that breast cancer will beresponsible for a woman's death is about 1 in 36 (about 3%). Death ratesfrom breast cancer have been declining since about 1989, with largerdecreases in women younger than 50. These decreases are believed to bethe result of earlier detection through screening and increasedawareness, as well as improved treatment.

The levels of pharmacodynamic markers can be assessed to determine theeffects of investigational agents, assaying tumors directly or surrogatetissues such as plasma. The ultimate goal is to incorporate predictivepharmacodynamic markers in early clinical studies of new oncology drugs.This would allow early evaluation of investigational agents based onhuman pharmacology data in a ‘real time’ setting. The biomarkersidentified herein can be used for cancer diagnostics, e.g., to predict(prior to treatment) and/or determine (after commencement of treatment)whether a cancer is resistant to a specific course of treatment. Thefollowing biomarkers were identified as valuable in the diagnosis andprediction of responsiveness to treatment for breast cancer: (i) totaland phosphorylated forms of Akt, MEK, mTOR, GSK3beta, and combinationsthereof, (ii) p70S6K, eIF4EBP1, PTEN, and (iii) combinations thereof.The invention includes the use of these biomarkers to indicate iftreatment with a therapeutic regimen targeting EGFR tyrosine kinaseactivity results in responsive or non-responsive outcomes.

Accordingly, the present invention includes a method and kit configuredto measure the levels of one or more of (i) total and phosphorylatedisoforms of Akt, MEK, mTOR, GSK3beta, and combinations thereof, (ii)p70S6K, eIF4EBP1, PTEN, and (iii) combinations thereof to determine if atumor is responsive and/or non-responsive to treatment with drugstargeting EGFR tyrosine kinase activity (e.g., gefitinib). In addition,the invention also relates to methods and kits to detect abnormal EGFRtyrosine kinase activity in a patient and/or to detect an alteration inEGFR tyrosine kinase activity by measuring one or more of the biomarkersidentified above and comparing those levels to a normal control and/or abaseline level to determine if an abnormality or alteration is presentin a patient sample.

Therefore, the invention provides a method for evaluating the efficacyof a treatment regimen in a patient diagnosed with breast cancer, saidmethod comprising (a) measuring a level of a biomarker in a test sampleobtained from a patient undergoing said treatment regimen for breastcancer, wherein said biomarker is selected from the group consisting of(i) total and phosphorylated isoforms of Akt, MEK, mTOR, GSK3beta, andcombinations thereof, (ii) p70S6K, eIF4EBP1, PTEN, and (iii)combinations thereof; and (b) evaluating from said level whether saidpatient is responsive to said treatment regimen.

The methods may include measuring a level of two or more biomarkers, ora panel of three or more such biomarkers. The panel may further compriseone or more additional biomarkers selected from HER2/neu, ER, PR, Ki67,and combinations thereof. Additional diagnostic methods can be used incombination with the panels described herein, as referenced in Misek etal, Int J. Proteomics, Vol. 2011, Article ID 343582, pages 1-9, thedisclosure of which is incorporated herein by reference in its entirety.

In one embodiment, the method includes measuring a level of a firstbiomarker and an additional biomarker, wherein the first biomarker is atotal form of a biomarker and the additional biomarker is aphosphorylated form of that biomarker. Diagnosis of the presence orstate of a cancer could be based on the absolute levels of one or bothof these forms. Alternatively, the diagnosis could be based on the ratioof phosphorylated to total forms (i.e., based on the fraction of aspecific biomarker that is present in a phosphorylated form). In oneexample, the method may include measuring a level of total Akt as thefirst biomarker and measuring a level of phosphorylated Akt as theadditional biomarker. Similarly, the method may comprise measuring oneor more of the following pairs of first and additional biomarkers, i.e.,total and phosphorylated biomarkers: total MEK and phosphorylated MEK;total GSK3β and phosphorylated GSK3β; and total mTOR and phosphorylatedmTOR.

The level(s) of the various biomarkers identified herein may reflect theresponsiveness or non-responsiveness of breast cancer to a giventreatment regimen. A response to a therapeutic regimen includes adetectable reduction to some extent of one or more of the symptoms ofbreast cancer, including, but not limited to: (1) reduction in thenumber of cancer cells; (2) reduction in tumor size; (3) inhibition(i.e., slowing to some extent, preferably stopping) of cancer cellinfiltration into peripheral organs; (4) inhibition (i.e., slowing tosome extent, preferably stopping) of tumor metastasis; (5) inhibition,to some extent, of tumor growth; (6) relieving or reducing to someextent one or more of the symptoms associated with the disorder; and/or(7) increasing, to some extent, the overall survival of a patientrelative to that observed for the standard of care for Breast cancer. Aresponse to a therapeutic regimen may also comprise maintenance of atherapeutic benefit, including, but not limited to (1) inhibiting anincrease in the number of cancer cells; (2) inhibiting an increase intumor size; (3) inhibiting cancer cell infiltration into peripheralorgans; (4) inhibiting tumor metastases; (5) relieving or reducing tosome extent one or more of the symptoms associated with the disorder;and/or (6) inhibiting a recurrence or onset of one or more of thesymptoms associated with the disorder.

The therapeutic regimen used in the method of the present invention mayinclude radiation treatment, chemotherapy, treatment with therapeuticdrugs, immune system modulation or other therapeutic regimens used incancer treatment. In one embodiment, the therapeutic regimen comprisesadministration of a therapeutic agent that modulates one or morebiological activities and the level(s) of said one or more biomarkersindicate modulation of said biological activities by said therapeuticagent. In particular, the biological activity includes EGFR tyrosinekinase activity and the therapeutic agent may be an agonist or anantagonist of such signaling pathway(s). In a preferred embodiment, thetherapeutic agent is gefitinib (Iressa), and it can be administeredalone or in combination with one or more additional therapeutic agents,e.g., trastuzumab, aromasin, anastrazole, taxotere (or another taxane),doxorubicin, adriamycin, Cytoxan, methotrexate, fluorouracil, andcombinations thereof.

The therapeutic regimen may include administration of a therapeuticagent or a combination of therapeutic agents to a patient one or moretimes over a given time period. For example, if the therapeutic agent isgefitinib, one suitable therapeutic regimen comprises administering thedrug once daily until the patient is no longer clinically benefitingfrom treatment or until unacceptable toxicity occurs. This treatmentregimen may be accompanied by the administration of one or moreadditional chemotherapeutic agents or palliative agents. The level(s) ofbiomarkers may be measured before treatment, one or more times duringthe administration period, and/or after treatment is suspended. Thelevel(s) of biomarkers may be measured at one or more time points in thetreatment regimen, e.g., before treatment, one or more times during thefour week administration period, and/or after the four weekadministration period. Therefore, the method may include measuring aninterim level of a biomarker during the therapeutic regimen and theevaluating step further comprises comparing that level, the interimlevel and the baseline level.

In addition, the level of a biomarker may be determined at any timepoint before and/or after initiation of treatment. In one embodiment,the biomarker is used to gauge the efficacy of a therapeutic regimen.Therefore, the method of the present invention may include measuring abaseline level(s) of a biomarker before a therapeutic regimen isinitiated, and the evaluating step further comprises comparing the leveland the baseline level. Moreover, the method may further comprisemeasuring an interim level of the biomarker during the therapeuticregimen and the evaluating step further comprises comparing the level,the interim level and the baseline level.

Alternatively, the measuring step may comprise measuring a level(s) of abiomarker before a therapeutic regimen is initiated to predict whetherbreast cancer will be responsive or non-responsive to a giventherapeutic regimen. The method may further comprise modifying thetherapeutic regimen based on the level(s) of a biomarker observed duringthe measuring step, e.g., increasing or decreasing the dosage,frequency, or route of administration of a therapeutic agent, adding anadditional therapeutic agent and/or palliative agent to a treatmentregimen, or if the therapeutic regimen includes the administration oftwo or more therapeutic and/or palliative agents, the treatment regimenmay be modified to eliminate one or more of the therapeutic and/orpalliative agents used in the combination therapy.

Still further, the evaluating step may include comparing the level of abiomarker to a detection cut-off level, wherein a level above thedetection cut-off level is indicative of breast cancer. Alternatively,the evaluating step comprises comparing a level of a biomarker to adetection cut-off level, wherein a level below the detection cut-offlevel is indicative of breast cancer.

In one embodiment of the present invention, the level of a biomarker iscompared to a detection cut-off level or range, wherein the biomarkerlevel above or below the detection cut-off level (or within thedetection cut-off range) is indicative of breast cancer. Furthermore,the levels of two or more biomarkers may both be used to make adetermination. For example, i) having a level of at least one of themarkers above or below a detection cut-off level (or within a detectioncut-off range) for that marker is indicative of breast cancer; ii)having the level of two or more (or all) of the markers above or below adetection cut-off level (or within a detection cut-off range) for eachof the markers is indicative of breast cancer; or iii) an algorithmbased on the levels of the multiple markers is used to determine ifbreast cancer is present.

As described herein, the measured levels of one or more biomarkers maybe used to detect or monitor cancer (e.g., breast cancer) and/or todetermine the responsiveness of a cancer to a specific treatmentregimen. The specific methods/algorithms for using biomarker levels tomake these determinations, as described herein, may optionally beimplemented by software running on a computer that accepts the biomarkerlevels as input and returns a report with the determinations to theuser. This software may run on a standalone computer or it may beintegrated into the software/computing system of the analytical deviceused to measure the biomarker levels or, alternatively, into alaboratory information management system (LIMS) into which crude orprocessed analytical data is entered. In one embodiment, biomarkers aremeasured in a point-of-care clinical device which carries out theappropriate methods/algorithms for detecting, monitoring or determiningthe responsiveness of a cancer and which reports such determination(s)back to the user.

In addition, the methods of the present invention may be used incombination with other methods of diagnosing breast cancer in a patient.In one embodiment, the patient may also be subjected to one or morediagnostic tools designed to detect breast cancer. For example, imagingmethods may be used to provide images of the breast to look for tumors.In addition, a biopsy may be performed. Imaging methods that may beperformed include ultrasound, computed tomography (CT) scan and magneticresonance imaging (MRI).

The assays of the present invention may be conducted by any suitablemethod. In one embodiment, the measuring step is conducted on a singlesample, and it may be conducted in a single assay chamber or assaydevice, including but not limited to a single well of an assay plate, asingle assay cartridge, a single lateral flow device, a single assaytube, etc.

According to one aspect of the invention, the level(s) of biomarker(s)are measured in samples collected from individuals clinically diagnosedwith, suspected of having or at risk of developing breast cancer.Initial diagnosis may have been carried out using conventional methods,e.g., biopsy or other conventional diagnostic methods. The level(s) ofbiomarker(s) are also measured in healthy individuals. Specificbiomarkers valuable in distinguishing between normal and diseasedpatients are identified by visual inspection of the data, for example,by visual classification of data plotted on a one-dimensional ormultidimensional graph, or by using statistical methods such ascharacterizing the statistically weighted difference between controlindividuals and diseased patients and/or by using Receiver OperatingCharacteristic (ROC) curve analysis. A variety of suitable methods foridentifying useful biomarkers and setting detectionthresholds/algorithms are known in the art and will be apparent to theskilled artisan.

For example and without limitation, diagnostically valuable biomarkersmay be first identified using a statistically weighted differencebetween control individuals and diseased patients, calculated as

$\frac{D - N}{\sqrt{\sigma_{D}*\sigma_{N}}}$

wherein D is the median level of a biomarker in patients diagnosed ashaving, for example, kidney cancer, N is the median (or average) of thecontrol individuals, σ_(D) is the standard deviation of D and σ_(N) isthe standard deviation of N. The larger the magnitude, the greater thestatistical difference between the diseased and normal populations.

According to one embodiment of the invention, biomarkers resulting in astatistically weighted difference between control individuals anddiseased patients of greater than, e.g., 1, 1.5, 2, 2.5 or 3 could beidentified as diagnostically valuable markers.

Another method of statistical analysis for identifying biomarkers is theuse of z-scores, e.g., as described in Skates et al. (2007) CancerEpidemiol. Biomarkers Prev. 16(2):334-341.

Another method of statistical analysis that can be useful in theinventive methods of the invention for determining the efficacy ofparticular candidate analytes, such as particular biomarkers, for actingas diagnostic marker(s) is ROC curve analysis. An ROC curve is agraphical approach to looking at the effect of a cut-off criterion,e.g., a cut-off value for a diagnostic indicator such as an assay signalor the level of an analyte in a sample, on the ability of a diagnosticto correctly identify positive or negative samples or subjects. One axisof the ROC curve is the true positive rate (TPR, i.e., the probabilitythat a true positive sample/subject will be correctly identified aspositive, or alternatively, the false negative rate (FNR=1-TPR, theprobability that a true positive sample/subject will be incorrectlyidentified as a negative). The other axis is the true negative rate,i.e., TNR, the probability that a true negative sample will be correctlyidentified as a negative, or alternatively, the false positive rate(FPR=1-TNR, the probability that a true negative sample will beincorrectly identified as positive). The ROC curve is generated usingassay results for a population of samples/subjects by varying thediagnostic cut-off value used to identify samples/subjects as positiveor negative and plotting calculated values of TPR or FNR and TNR or FPRfor each cut-off value. The area under the ROC curve (referred to hereinas the AUC) is one indication of the ability of the diagnostic toseparate positive and negative samples/subjects. In one embodiment, abiomarker provides an AUC≧0.7. In another embodiment, a biomarkerprovides an AUC≧0.8. In another embodiment, a biomarker provides an AUC0.9.

Diagnostic indicators analyzed by ROC curve analysis may be a level ofan analyte, e.g., a biomarker, or an assay signal. Alternatively, thediagnostic indicator may be a function of multiple measured values, forexample, a function of the level/assay signal of a plurality ofanalytes, e.g., a plurality of biomarkers, or a function that combinesthe level or assay signal of one or more analytes with a patient'sscoring value that is determined based on visual, radiological and/orhistological evaluation of a patient. The multi-parameter analysis mayprovide more accurate diagnosis relative to analysis of a single marker.

Candidates for a multi-analyte panel could be selected by using criteriasuch as individual analyte ROC areas, median difference between groupsnormalized by geometric interquartile range (IQR) etc. The objective isto partition the analyte space to improve separation between groups (forexample, normal and disease populations) or to minimize themisclassification rate.

One approach is to define a panel response as a weighted combination ofindividual analytes and then compute an objective function like ROCarea, product of sensitivity and specificity, etc. See e.g., WO2004/058055, as well as US2006/0205012, the disclosures of which areincorporated herein by reference in their entireties.

Biomarker levels may be measured using any of a number of techniquesavailable to the person of ordinary skill in the art, e.g., directphysical measurements (e.g., mass spectrometry) or binding assays (e.g.,immunoassays, agglutination assays and immunochromatographic assays).The method may also comprise measuring a signal that results from achemical reactions, e.g., a change in optical absorbance, a change influorescence, the generation of chemiluminescence orelectrochemiluminescence, a change in reflectivity, refractive index orlight scattering, the accumulation or release of detectable labels fromthe surface, the oxidation or reduction or redox species, an electricalcurrent or potential, changes in magnetic fields, etc. Suitabledetection techniques may detect binding events by measuring theparticipation of labeled binding reagents through the measurement of thelabels via their photoluminescence (e.g., via measurement offluorescence, time-resolved fluorescence, evanescent wave fluorescence,up-converting phosphors, multi-photon fluorescence, etc.),chemiluminescence, electrochemiluminescence, light scattering, opticalabsorbance, radioactivity, magnetic fields, enzymatic activity (e.g., bymeasuring enzyme activity through enzymatic reactions that cause changesin optical absorbance or fluorescence or cause the emission ofchemiluminescence). Alternatively, detection techniques may be used thatdo not require the use of labels, e.g., techniques based on measuringmass (e.g., surface acoustic wave measurements), refractive index (e.g.,surface plasmon resonance measurements), or the inherent luminescence ofan analyte.

Binding assays for measuring biomarker levels may use solid phase orhomogenous formats. Suitable assay methods include sandwich orcompetitive binding assays. Examples of sandwich immunoassays aredescribed in U.S. Pat. No. 4,168,146 and U.S. Pat. No. 4,366,241, bothof which are incorporated herein by reference in their entireties.Examples of competitive immunoassays include those disclosed in U.S.Pat. No. 4,235,601, U.S. Pat. No. 4,442,204 and U.S. Pat. No. 5,208,535,each of which are incorporated herein by reference in their entireties.

Multiple biomarkers may be measured using a multiplexed assay format,e.g., multiplexing through the use of binding reagent arrays,multiplexing using spectral discrimination of labels, multiplexing offlow cytometric analysis of binding assays carried out on particles,e.g., using the Luminex® system. Suitable multiplexing methods includearray based binding assays using patterned arrays of immobilizedantibodies directed against the biomarkers of interest. Variousapproaches for conducting multiplexed assays have been described (Seee.g., US 20040022677; US 20050052646; US 20030207290; US 20030113713; US20050142033; and US 20040189311, each of which is incorporated herein byreference in their entireties. One approach to multiplexing bindingassays involves the use of patterned arrays of binding reagents, e.g.,U.S. Pat. Nos. 5,807,522 and 6,110,426; Delehanty J-B., Printingfunctional protein microarrays using piezoelectric capillaries, MethodsMol. Bio. (2004) 278: 135-44; Lue R Y et al., Site-specificimmobilization of biotinylated proteins for protein microarray analysis,Methods Mol. Biol. (2004) 278: 85-100; Lovett, Toxicogenomics:Toxicologists Brace for Genomics Revolution, Science (2000) 289:536-537; Berns A, Cancer: Gene expression in diagnosis, nature (2000),403,491-92; Walt, Molecular Biology: Bead-based Fiber-Optic Arrays,Science (2000) 287: 451-52 for more details). Another approach involvesthe use of binding reagents coated on beads that can be individuallyidentified and interrogated. See e.g., WO 9926067, which describes theuse of magnetic particles that vary in size to assay multiple analytes;particles belonging to different distinct size ranges are used to assaydifferent analytes. The particles are designed to be distinguished andindividually interrogated by flow cytometry. Vignali has described amultiplex binding assay in which 64 different bead sets ofmicroparticles are employed, each having a uniform and distinctproportion of two dyes (Vignali, D. A A, “Multiplexed Particle-BasedFlow Cytometric Assays” J. ImmunoL Meth. (2000) 243: 243-55). A similarapproach involving a set of 15 different beads of differing size andfluorescence has been disclosed as useful for simultaneous typing ofmultiple pneumococcal serotypes (Park, M. K et al., “A Latex Bead-BasedFlow Cytometric Immunoassay Capable of Simultaneous Typing of MultiplePneumococcal Serotypes (Multibead Assay)” Olin. Diag. Lab ImmunoL (2000)7: 4869). Bishop, J E et al. have described a multiplex sandwich assayfor simultaneous quantification of six human cytokines (Bishop, L E. etal., “Simultaneous Quantification of Six Human Cytokines in a SingleSample Using Microparticle-based Flow Cytometric Technology,” Olin. Chem(1999) 45:1693-1694).

A diagnostic test may be conducted in a single assay chamber, such as asingle well of an assay plate or an assay chamber that is an assaychamber of a cartridge. The assay modules, e.g., assay plates orcartridges or multi-well assay plates), methods and apparatuses forconducting assay measurements suitable for the present invention aredescribed for example, in US 20040022677; US 20050052646; US20050142033; US 20040189311, each of which is incorporated herein byreference in their entireties. Assay plates and plate readers are nowcommercially available (MULTI-SPOT® and MULTI-ARRAY® plates and SECTOR®instruments, Meso Scale Discovery, a division of Meso Scale Diagnostics,LLC, Rockville, Md.).

The present invention relates to a kit for the analysis of a panel oftarget analytes. The kit is preferably configured to conduct amultiplexed assay of two or more of the following analytes: (i) totaland phosphorylatedisoforms of Akt, MEK, mTOR, GSK3beta, and combinationsthereof, (ii) p70S6K, eIF4EBP1, PTEN, and (iii) combinations thereof.The kit can include (a) a single panel arrayed on a multi-well platewhich is configured to be used in an electrochemiluminescence assay, aswell as (b) associated consumables, e.g., detection antibodies,calibrators, and optional diluents and/or buffers. Alternatively, themulti-well plates and associated consumables can be provided separately.

The panel is preferably configured in a multi-well assay plate includinga plurality of wells, each well having an array with “spots” or discretebinding domains. Preferably, the array includes one, four, seven, ten,sixteen, or twenty-five binding domains, and most preferably, the arrayincludes one, four, seven, or ten binding domains. A capture antibody toeach analyte is immobilized on a binding domain in the well and thatcapture antibody is used to detect the presence of the target analyte inan immunoassay. Briefly, a sample suspected of containing that analyteis added to the well and if present, the analyte binds to the captureantibody at the designated binding domain. The presence of bound analyteon the binding domain is detected by adding labeled detection antibody.The detection antibody also binds to the analyte forming a “sandwich”complex (capture antibody-analyte-detection antibody) on the bindingdomain.

The multiplexed immunoassay kits described herein allow a user tosimultaneously quantify multiple biomarkers. The panels are selected andoptimized such that the individual assays function well together. Thesample may require dilution prior to being assayed. Sample dilutions forspecific sample matrices of interest are optimized for a given panel tominimize sample matrix effects and to maximize the likelihood that allthe analytes in the panel will be within the dynamic range of the assay.In a preferred embodiment, all of the analytes in the panel are analyzedwith the same sample dilution in at least one sample type. In anotherpreferred embodiment, all of the analytes in a panel are measured usingthe same dilution for most sample types.

For a given panel, the detection antibody concentration and the numberof labels per protein (UP ratio) for the detection antibody are adjustedto bring the expected levels of all analytes into a quantifiable rangeat the same sample dilution. If one wants to increase the high end ofthe quantifiable range for a given analyte, then the L/P can bedecreased and/or the detection antibody concentration is decreased. Onthe other hand, if one wants to increase the lower end of thequantifiable range, the L/P can be increased, the detection antibodyconcentration can be increased if it is not at the saturation level,and/or the background signal can be lowered.

Calibration standards for use with the assay panels are selected toprovide the appropriate quantifiable range with the recommended sampledilution for the panel. The calibration standards have knownconcentrations of one of more of the analytes in the panel.Concentrations of the analytes in unknown samples are determined bycomparison to these standards. In one embodiment, calibration standardscomprise mixtures of the different analytes measured by an assay panel.Preferably, the analyte levels in a combined calibrator are selectedsuch that the assay signals for each analyte are comparable, e.g.,within a factor of two, a factor of five or a factor of 10. In anotherembodiment, calibration standards include mixtures of analytes frommultiple different assay panels.

A calibration curve may be fit to the assay signals measured withcalibration standards using, e.g., curve fits known in the art such aslinear fits, 4-parameter logistic (4-PL) and 5-parameter (5-PL) fits.Using such fits, the concentration of analytes in an unknown sample maybe determined by backfitting the measured assay signals to thecalculated fits. Measurements with calibration standards may also beused to determine assay characteristics such as the limit of detection(LOD), limit of quantification (LOQ), dynamic range, and limit oflinearity (LOL).

A kit can include the following assay components: a multi-well assayplate configured to conduct an immunoassay for one of the panelsdescribed herein, a set of detection antibodies for the analytes in thepanel (wherein the set comprises individual detection antibodies and/ora composition comprising a blend of one or more individual detectionantibodies), and a set of calibrators for the analytes in the panel(wherein the set comprises individual calibrator protein compositionsand/or a composition comprising a blend of one or more individualcalibrator proteins). The kit can also include one of more of thefollowing additional components: a blocking buffer (used to block assayplates prior to addition of sample), an antibody diluent (used to dilutestock detection antibody concentrations to the working concentration),an assay diluent (used to dilute samples), a calibrator diluent (used todilute or reconstitute calibration standards) and a read buffer (used toprovide the appropriate environment for detection of assay labels, e.g.,by an ECL measurement). The antibody and assay diluents are selected toreduce background, optimize specific signal, and reduce assayinterference and matrix effect. The calibrator diluent is optimized toyield the longest shelf life and retention of calibrator activity. Theblocking buffer should be optimized to reduce background. The readbuffer is selected to yield the appropriate sensitivity, quantifiablerange, and slowest off-rate.

The reagent components of the kit can be provided as liquid reagents,lyophilized, or combinations thereof, diluted or undiluted, and the kitincludes instructions for appropriate preparation of reagents prior touse. In a preferred embodiment, a set of detection antibodies areincluded in the kit comprising a plurality of individual detectionantibody compositions in liquid form. Moreover, the set of calibratorsprovided in the kit preferably comprise a lyophilized blend ofcalibrator proteins. Still further, the kit includes a multi-well assayplate that has been pre-coated with capture antibodies and exposed to astabilizing treatment to ensure the integrity and stability of theimmobilized antibodies.

As part of a multiplexed panel development, assays are optimized toreduce calibrator and detection antibody non-specific binding. Insandwich immunoassays, specificity mainly comes from capture antibodybinding. Some considerations for evaluating multiplexed panels include:(a) detection antibody non-specific binding to capture antibodies isreduced to lower background of assays in the panel, and this can beachieved by adjusting the concentrations and LIP of the detectionantibodies; (b) non-specific binding of detection antibodies to othercalibrators in the panel is also undesirable and should be minimized;(c) non-specific binding of other calibrators in the panel and otherrelated analytes should be minimized; if there is calibratornon-specific binding, it can reduce the overall specificity of theassays in the panel and it can also yield unreliable results as therewill be calibrator competition to bind the capture antibody.

Different assays in the panel may require different incubation times andsample handling requirements for optimal performance. Therefore, thegoal is to select a protocol that's optimized for most assays in thepanel. Optimization of the assay protocol includes, but is not limitedto, adjusting one or more of the following protocol parameters: timing(incubation time of each step), preparation procedure (calibrators,samples, controls, etc.), and number of wash steps.

The reagents used in the kits, e.g., the detection and captureantibodies and calibrator proteins, are preferably subjected toanalytical testing and meet or exceed the specifications for thosetests. The analytical tests that can be used to characterize kitmaterials include but are not limited to, CIEF, DLS, reducing and/ornon-reducing EXPERION, denaturing SOS-PAGE, non-denaturing SOS-PAGE,SEC-MALS, and combinations thereof. In a preferred embodiment, thematerials are characterized by CIEF, DLS, and reducing and non-reducingEXPERION. One or more additional tests, including but not limited todenaturing SDS-PAGE, non-denaturing SOS-PAGE, SEC-MALS, and combinationsthereof, can also be used to characterize the materials. In a preferredembodiment, the materials are also subjected to functional testing,i.e., a binding assay for the target analyte, as well as one or morecharacterization tests, such as those listed above. If the materials donot meet or exceed the specifications for the functional and/orcharacterization tests, they can be subjected to additional purificationsteps and re-tested. Each of these tests and the metrics applied to theanalysis of raw materials subjected to these tests are described below:

Capillary Isoelectric Focusing (CIEF) is a technique commonly used toseparate peptides and proteins, and it is useful in the detection ofaggregates. During a CIEF separation, a capillary is filled with thesample in solution and when voltage is applied, the ions migrate to aregion where they become neutral (pH=pl). The anodic end of thecapillary sits in acidic solution (low pH), while the cathodic end sitsin basic solution (high pH). Compounds of equal isoelectric points (pl)are “focused” into sharp segments and remain in their specific zone,which allows for their distinct detection based on molecular charge andisoelectric point. Each specific antibody solution will have afingerprint CIEF that can change over time. When a protein solutiondeteriorates, the nature of the protein and the charge distribution canchange. Therefore, CIEF is a particularly useful tool to assess therelative purity of a protein solution and it is a preferred method ofcharacterizing the antibodies and calibrators in the plates and kitsdescribed herein. The metrics used in CIEF include pl of the main peak,the pl range of the solution, and the profile shape, and each of thesemeasurements are compared to that of a reference standard.

Dynamic Light Scattering (DLS) is used to probe the diffusion ofparticulate materials either in solution or in suspension. Bydetermining the rate of diffusion (the diffusion coefficient),information regarding the size of particles, the conformation ofmacromolecular chains, various interactions among the constituents inthe solution or suspension, and even the kinetics of the scatterers canbe obtained without the need for calibration. In a DLS experiment, thefluctuations (temporal variation, typically in a μs to ms time scale) ofthe scattered light from scatterers in a medium are recorded andanalyzed in correlation delay time domain. Like CIEF, each proteinsolution will generate a fingerprint DLS for the particle size and it'sideally suited to detect aggregation. All IgGs, regardless of bindingspecificity, will exhibit the same DLS particle size. The metrics usedto analyze a protein solution using DLS include percentagepolydispersity, percentage intensity, percentage mass, and the radius ofthe protein peak. In a preferred embodiment, an antibody solution meetsor exceeds one or more of the following DLS specifications: (a) radiusof the antibody peak: 4-8 nm (antibody molecule size); (b)polydispersity of the antibody peak: <40% (measure of size heterogeneityof antibody molecules); (c) intensity of the antibody peak: >50% (ifother peaks are present, then the antibody peak is the predominantpeak); and (d) mass in the antibody peak: >50%.

Reducing and non-reducing gel electrophoresis are techniques well knownin the art. The EXPERION™ (Bio-Rad Laboratories, Inc., www.bio-rad.com)automated electrophoresis station performs all of the steps of gel-basedelectrophoresis in one unit by automating and combining electrophoresis,staining, destaining, band detection, and imaging into a single step. Itcan be used to measure purity. Preferably, an antibody preparation isgreater 50% pure by Experion, more preferably, greater than 75% pure,and most preferably greater than 80% pure. Metrics that are applied toprotein analysis using non-reducing Experion include percentage totalmass of protein, and for reducing Experion they include percentage totalmass of the heavy and light chains in an antibody solution, and theheavy to light chain ratio.

Multi-Angle Light Scattering (MALS) detection can be used in thestand-alone (batch) mode to measure specific or non-specific proteininteractions, as well as in conjunction with a separation system such asflow field flow fractionation (FFF) or size exclusion chromatography(SEC). The combined SEC-MALS method has many applications, such as theconfirmation of the oligomeric state of a protein, quantification ofprotein aggregation, and determination of protein conjugatestoichiometry. Preferably, this method is used to detect molecularweight of the components of a sample.

As used herein, a lot of kits comprise a group of kits comprising kitcomponents that meet a set of kit release specifications. A lot caninclude at least 10, at least 100, at least 500, at least 1,000, atleast 5,000, or at least 10,000 kits and a subset of kits from that lotare subjected to analytical testing to ensure that the lot meets orexceeds the release specifications. In one embodiment, the releasespecifications include but are not limited to kit processing, reagentstability, and kit component storage condition specifications. Kitprocessing specifications include the maximum total sample incubationtime and the maximum total time to complete an assay using the kit.Reagent stability specifications include the minimum stability of eachreagent component of the kit at a specified storage temperature. Kitstorage condition specifications include the range of storagetemperatures for all components of the kit, the maximum storagetemperature for frozen components of the kit, and the maximum storagetemperature for non-frozen components of the kit. A subset of kits in alot is reviewed in relation to these specifications and the size of thesubset depends on the lot size. In a preferred embodiment, for a lot ofup to 300 kits, a sampling of 4-7 kits are tested; for a lot of 300-950kits, a sampling of 8-10 kits are tested; and for a lot of greater than950 kits, a sampling of 10-12 kits are tested. Alternatively oradditionally, a sampling of up to 1-5% preferably up to 1-3%, and mostpreferably up to 2% is tested.

In addition, each lot of multi-well assay plates is preferably subjectedto uniformity and functional testing. A subset of plates in a lot issubjected to these testing methods and the size of the subset depends onthe lot size. In a preferred embodiment, for a lot of up to 300 plates,a sampling of 4-7 plates are tested; for a lot of 300-950 plates, asampling of 8-10 plates are tested; and for a lot of greater than 950plates, a sampling of 10-12 plates are tested. Alternatively oradditionally, a sampling of up to 1-5% preferably up to 1-3%, and mostpreferably up to 2% is tested. The uniformity and functional testingspecifications are expressed in terms of % CV, Coefficient ofVariability, which is a dimensionless number defined as the standarddeviation of a set of measurements, in this case, the relative signaldetected from binding domains across a plate, divided by the mean of theset.

One type of uniformity testing is protein A/G testing. Protein A/Gbinding is used to confirm that all binding domains within a plate arecoupled to capture antibody. Protein A/G is a recombinant fusion proteinthat combines IgG binding domains of Protein A and protein G and itbinds to all subclasses of human IgG, as well as IgA, IgE, IgM and, to alesser extent, IgD. Protein A/G also binds to all subclasses of mouseIgG but not mouse IgA, IgM, or serum albumin, making it particularlywell suited to detect mouse monoclonal IgG antibodies withoutinterference from IgA, IgM, and serum albumin that might be present inthe sample matrix. Protein A/G can be labeled with a detectable moiety,e.g., a fluorescent, chemiluminescent, or electrochemiluminescent label,preferably an ECL label, to facilitate detection. Therefore, if captureantibody is adhered to a binding domain of a well, it will bind tolabeled protein A/G, and the relative amount of capture antibody boundto the surface across a plate can be measured.

In addition to the uniformity testing described above, a uniformitymetric for a subset of plates within a lot can be calculated to assesswithin-plate trending. A uniformity metric is calculated using a matrixof normalized signals from protein A/G and/or other uniformity orfunctional tests. The raw signal data is smoothed by techniques known inthe art, thereby subtracting noise from the raw data, and the uniformitymetric is calculated by subtracting the minimum signal in the adjusteddata set from the maximum signal.

In a preferred embodiment, a subset of plates in a lot is subjected toprotein A/G and functional testing and that subset meet or exceed thefollowing specifications:

TABLE 3(a) Plate Metrics Preferred Specification for a subset of 96 wellMetric multi-well plates Average intraplate CV ≦10% Maximum intraplateCV ≦13% Average Uniformity ≦25% Maximum Uniformity ≦37% CV of intraplateaverages ≦18% Signal, lower boundary >1500 Signal, upper boundary <10⁽⁶⁾

As disclosed in U.S. Pat. No. 7,842,246 to Wohlstadter et al., thedisclosure of which is incorporated herein by reference in its entirety,each plate consists of several elements, e.g., a plate top, a platebottom, wells, working electrodes, counter electrodes, referenceelectrodes, dielectric materials, electrical connects, and assayreagents. The wells of the plate are defined by holes/openings in theplate top. The plate bottom can be affixed, manually or by automatedmeans, to the plate top, and the plate bottom can serve as the bottom ofthe well. Plates may have any number of wells of any size or shape,arranged in any pattern or configuration, and they can be composed of avariety of different materials. Preferred embodiments of the inventionuse industry standard formats for the number, size, shape, andconfiguration of the plate and wells. Examples of standard formatsinclude 96, 384, 1536, and 9600 well plates, with the wells configuredin two-dimensional arrays. Other formats may include single well plates(preferably having a plurality of assay domains that form spot patternswithin each well), 2 well plates, 6 well plates, 24 well plates, and6144 well plates. Each well of the plate includes a spot pattern ofvarying density, ranging from one spot within a well to 2, 4, 7, 9, 10,16, 25, etc., as described hereinabove.

Each plate is assembled according to a set of preferred specifications.In a preferred embodiment, a plate bottom meets or exceeds the followingspecifications:

TABLE 3(b) Plate bottom specifications 96-well (round well)specifications in Parameter inches Length range (C to C)* 3.8904-3.9004(A1-A12 and H1-H12)** Width range (C to C) 2.4736-2.4836 (A1-A12 andH1-H12) Well to well spacing 0.3513-0.3573 *C to C well distance is thecenter of spot to center of spot distance between the outermost wells ofa plate.

In a further preferred embodiment, the plate also meets or exceedsdefined specifications for alignment of a spot pattern within a well ofthe plate. These specifications include three parameters: (a) Δx, thedifference between the center of the spot pattern and the center of thewell along the x axis of the plate (column-wise, long axis); (b) Δy, thedifference between the center of the spot pattern and the center of thewell along the y axis of the plate (row-wise, short axis); and (c) α,the counter-clockwise angle between the long axis of the plate bottomand the long axis of the plate top of a 96-well plate. In a preferredembodiment, the plate meets or exceeds the following specifications:Δx≦0.2 mm, Δy≦0.2 mm, and α≦0.1°.

The following non-limiting examples serve to illustrate rather thanlimit the present invention.

Examples Measurement of Biomarkers Indicative of Gefitinib Sensitivityin the Treatment of Breast Cancer

Multiplex immunoassay kits were used for detection of total and/orphosphorylated biomarkers (supplied by Meso Scale Diagnostics, LLC.,Rockville, Md.). Levels of each biomarker were determined by calibrationof the assays with were either purified calibrator proteins or usingcontrol cell lysates from appropriately treated cultured cell lines(e.g., cells subjected to conditions known to induce or reduce levels ofa specific biomarker). Calibration curves were derived by testing serialdilutions of the calibrator lysates or purified proteins. Levels ofbiomarkers in test samples were back-calculated from the calibrationcurves and were expressed in terms of wt. (or arbitrary units) ofprotein per weight of tissue extract (for purified calibrators) or interms of weight of crude control lysate protein per well (for lysatecalibrators). Titrations of tumor extracts were carried out to determinethe linearity of the assay response to sample dilution and to select thesample dilution that would be appropriate to use for each assay panel.

The following cell lines were used, representing different breast cancersubtypes and having known differences in sensitivity to gefitinib asindicated by their IC50 values. Cells were treated with 1 uM gefitinib.

TABLE 4 Cell Line Sub-type IC50 (μM) HER2 ER MDA-MB-175 Luminal 0.03 − +BT-474 HER2 amplified 0.13 + − HCC1954 HER2 amplified 0.23 + − SKBR3HER2 amplified 0.34 + − SUM190 HER2 amplified 0.38 + − MDA-MB-468Basal/EGFR amplified 0.78 − − UACC812 HER2 amplified 1.25 + − T47DLuminal 1.7 − + CAL 51 Basal/vimentin+ 3.3 − − MDA-MB-453 HER2 amplified5.7 + − ZR75-1 Luminal 7 − + HCC1937 Basal 10 − − EFM19 Luminal 10 − +BT20 Basal >10 − − MCF7 Luminal >10 − + MDA-MB-231 Basal/vimentin+ >10 −− CAMA1 Luminal >10 − +

In general, the assay format was as follows, with minor alterations forspecific assay panels as indicated in the assay protocols provided witheach assay kit (supplied by Meso Scale Diagnostics, LLC): (1) block MSDMULTI-SPOT® plate for 1 hour with appropriate MSD® blocking solution andwash; (2) add 25 μl assay diluent to each well, if specified; (3) add 25μl calibrator, or sample (diluted as appropriate) to each well; (4)incubate with shaking for 1-3 hours (time as specified) and wash thewell; (5) add 25 μl labeled detection antibody solution to each well;(6) incubate with shaking for 1-2 hours (time as specified) and wash thewell; (7) add 150 pl MSD read buffer to each well; (8) read plateimmediately on MSD 516000 Reader (supplied by Meso Scale Diagnostics,LLC).

Fractional marker levels (to 0 h time point) for a representative set ofbiomarkers is shown in FIG. 1. The levels of several markers (e.g.,phospho-Akt, phospho-GSK3beta, phospho-MEK, and phospho-mTOR, weredecreased correlating with IC50 values.

The fractional levels of each biomarker (relative to the t=0 h values)in each cell line were correlated to the known gefitinib IC50 values forthe cells lines. Table 5 summarizes the correlation coefficients foreach of the markers at each of the time points analyzed across all ofthe cell lines' IC50 values. The sums of the correlation coefficientsfor each marker suggest which markers change in levels over multipletime points indicating a persistent response to drug. The top fourmarkers were found to be phospho-Akt, phospho-GSK3beta, phospho-MEK, andphospho-mTOR.

TABLE 5 Correlation coefficients for changes in levels of each markerversus IC50 values for the 17 breast cancer cell lines evaluated atindividual time points. The sums of the correlation coefficients foreach marker are shown. p- p- p- p- p- p- p-Akt MEK GSK3? mTOR mTORp70S6K MEK 4EBP1 Akt EGFR IGF1R p70S6K VEGF 10 min 0.70 0.59 0.49 0.24−0.16   0.36 −0.14 0.31 −0.05 −0.18 0.01 −0.10   0.19 0.5 h  0.75 0.710.54 0.48 0.35 0.50   0.31 0.29   0.43   0.41 0.20   0.18   0.60  1 h0.79 0.62 0.63 0.66 0.59 0.56   0.47 0.58   0.17   0.63 0.50   0.20  0.37  8 h 0.79 0.57 0.69 0.65 0.44 0.55   0.55 0.34   0.32   0.50 0.25  0.14   0.24 24 h 0.71 0.56 0.56 0.51 0.42 0.21   0.41 0.04   0.47−0.19 0.24   0.38   0.17 48 h 0.64 0.62 0.58 0.61 0.78 0.16   0.57 0.51  0.69   0.57 0.47   0.75 −0.01 SUM 4.38 3.68 3.49 3.16 2.43 2.35   2.172.07   2.04   1.73 1.67   1.55   1.55 p- VEGFR2 PTEN Raptor GSK3? RictorPTEN EGFR ERK PI3K p-IGF1R 4EBP1 cMet 10 min 0.23 0.02 0.06 −0.41 −0.19−0.20 −0.23 0.16 −0.37 0.07 −0.13 −0.30 0.5 h  0.48 0.17 −0.01 −0.370.18 −0.02 −0.01 0.03 −0.45 0.13 −0.02   0.07  1 h 0.04 0.41 −0.05 0.290.10 0.38 0.24 0.13 0.06   0.07   0.04  8 h 0.22 0.25 0.07 0.15 −0.120.04 −0.06 −0.26 0.25 −0.20 −0.36   0.07 24 h 0.21 0.23 0.00 0.27 0.010.00 −0.18 −0.05 −0.20 −0.09 −0.41 48 h 0.12 0.17 0.62 0.52 0.31 0.000.21 −0.17 0.25 −0.09   0.25   0.15 SUM 1.30 1.24 0.69 0.47 0.30 0.21−0.02 −0.16 −0.26 −0.29 −0.29 −0.38

The relative levels of optimal biomarkers as a function of IC50 valuesfor the 1 hour time point are illustrated in FIGS. 2(a)-(d) (cut off forsensitivity is 1 uM). Each point on the graphs represents a cell line.The relative levels of the combined markers phospho-Akt,phospho-GSK3beta, phospho-MEK, and phospho-mTOR as a function of IC50values for the 1 hour time point is illustrated in FIG. 3. Each point onthe graph represents the sum of the relative levels of each marker shownin FIG. 2(a)-(d) for one cell line as a function of its 1050 value forgefitinib.

Various publications and test methods are cited herein, the disclosuresof which are incorporated herein by reference in their entireties, Incases where the present specification and a document incorporated byreference and/or referred to herein include conflicting disclosure,and/or inconsistent use of terminology, and/or theincorporated/referenced documents use or define terms differently thanthey are used or defined in the present specification, the presentspecification shall control.

1. A method for evaluating the efficacy of a treatment regimen in apatient diagnosed with breast cancer, said method comprising (a)obtaining a test sample from a patient undergoing said treatment regimenfor breast cancer; (b) measuring a level of a biomarker in said testsample, wherein said biomarker comprises (i) total and phosphorylatedisoforms of Akt, MEK, mTOR, GSK3beta, and combinations thereof, (ii)p70S6K, eIF4EBP1, PTEN, and (iii) combinations thereof; (c) comparingsaid level to a normal control level of said biomarker; and (d)evaluating from said comparing step (c) whether said patient isresponsive to said treatment regimen.
 2. A method for evaluating theefficacy of a treatment regimen in a patient diagnosed with breastcancer, said method comprising (a) ordering a test comprising ameasurement of a level of a biomarker in a test sample obtained from apatient undergoing said treatment regimen for breast cancer, whereinsaid biomarker comprises (i) total and phosphorylated isoforms of Akt,MEK, mTOR, GSK3beta, and combinations thereof, (ii) p70S6K, eIF4EBP1,PTEN, and (iii) combinations thereof; (b) comparing said level to anormal control level of said biomarker; and (c) evaluating from saidcomparing step (b) whether said patient is responsive to said treatmentregimen.
 3. A method of administering a treatment regimen to a patientin need thereof for treating breast cancer, comprising: (a) obtaining atest sample from a patient undergoing said treatment regimen for breastcancer; (b) measuring a level of a biomarker in said test sample,wherein said biomarker comprises (i) total and phosphorylated isoformsof Akt, MEK, mTOR, GSK3beta, and combinations thereof, (ii) p70S6K,eIF4EBP1, PTEN, and (iii) combinations thereof; (c) comparing said levelto a normal control level of said biomarker; (d) evaluating from saidcomparing step (c) whether said patient is responsive to said treatmentregimen; and (e) adjusting said treatment regimen based on saidevaluating step (d).
 4. A method of administering a treatment regimen toa patient in need thereof for treating breast cancer, comprising: (a)obtaining a test sample from a patient prior to the commencement of saidtreatment regimen for breast cancer; (b) measuring a level of abiomarker in said test sample, wherein said biomarker comprises (i)total and phosphorylated isoforms of Akt, MEK, mTOR, GSK3beta, andcombinations thereof, (ii) p70S6K, eIF4EBP1, PTEN, and (iii)combinations thereof; (c) comparing said level to a normal control levelof said biomarker; (d) evaluating from said comparing step (c) whethersaid patient will be responsive to said treatment regimen; and (e)administering said treatment regimen based on said evaluating step (d).5. A method of administering a treatment regimen to a patient in needthereof for treating breast cancer, comprising: (a) evaluating a levelof a biomarker in a test sample obtained from a patient undergoing saidtreatment regimen for breast cancer relative to a normal control levelof said biomarker, wherein said biomarker comprises (i) total andphosphorylated isoforms of Akt, MEK, mTOR, GSK3beta, and combinationsthereof, (ii) p70S6K, eIF4EBP1, PTEN, and (iii) combinations thereof;and (b) adjusting said treatment regimen based on said evaluating step(a).
 6. A method of administering a treatment regimen to a patient inneed thereof for treating breast cancer, comprising: (a) evaluating alevel of a biomarker in a test sample obtained from a patient prior tothe commencement of said treatment regimen for breast cancer relative toa normal control level of said biomarker, wherein said biomarkercomprises (i) total and phosphorylated isoforms of Akt, MEK, mTOR,GSK3beta, and combinations thereof, (ii) p70S6K, eIF4EBP1, PTEN, and(iii) combinations thereof; and (b) administering said treatment regimenbased on said evaluating step (a).
 7. A method of treating breastcancer, comprising administering a VEGF-inhibitor to a patient whoselevels of a biomarker for RCC or metastatic melanoma were determined tobe at least 1.5-fold greater/less than those values in a normal control,wherein said biomarker of breast cancer is selected from (i) total andphosphorylated isoforms of Akt, MEK, mTOR, GSK3beta, and combinationsthereof, (ii) p70S6K, eIF4EBP1, PTEN, and (iii) combinations thereof. 8.The method of claim 1 wherein said measuring step comprises conducting amultiplexed assay measurement of a plurality of said biomarkers in saidtest sample, wherein said multiplexed assay measurement is conductedusing one reaction volume comprising said test sample.
 9. The method ofclaim 1 wherein said method comprises measuring levels of two or morebiomarkers.
 10. The method of claim 9 wherein said measuring stepcomprises measuring levels of a first biomarker and an additionalbiomarker, wherein said first biomarker is a total form of a biomarkerand said additional biomarker is a phosphorylated form of saidbiomarker.
 11. The method of claim 1, wherein said treatment regimencomprises administration of a therapeutic agent comprising gefitinib.12. The method of claim 1 further comprising one or more additionalmeasuring steps including: (x) measuring a baseline level(s) of saidbiomarker before said treatment regimen is initiated, and saidevaluating step further comprises comparing said level and said baselinelevel; and (y) measuring an interim level of said biomarker during saidtreatment regimen and said evaluating step further comprises comparingsaid level, said interim level and said baseline level.
 13. The methodof claim 1, wherein said evaluating step comprises comparing said levelof said biomarker to a detection cut-off level, wherein said level abovesaid detection cut-off level is indicative of breast cancer.
 14. Themethod of claim 1, wherein said evaluating step comprises comparing saidlevel of said biomarker to a detection cut-off level, wherein said levelbelow said detection cut-off level is indicative of breast cancer. 15.The method of claim 1 further comprising determining from said level ofsaid biomarker the disease progression of breast cancer. 16.-22.(canceled)